CN102692502B - Rapid hepatitis A virus IgM antibody detection kit and preparation method thereof - Google Patents
Rapid hepatitis A virus IgM antibody detection kit and preparation method thereof Download PDFInfo
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- CN102692502B CN102692502B CN201210125222.7A CN201210125222A CN102692502B CN 102692502 B CN102692502 B CN 102692502B CN 201210125222 A CN201210125222 A CN 201210125222A CN 102692502 B CN102692502 B CN 102692502B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a rapid hepatitis A virus IgM antibody detection kit which is realized by using reaction principles of a capture method and combinedly using colloidal gold immunochromatographic assay technology. The kit comprises anti-human IgM, antigen antibody complexes coupled with colloidal gold, supportive materials and a quality control reaction system. According to a technical scheme, anti-human IgM is coated on a detection line (T). The virus antigen antibody complexes are coupled with the colloidal gold and coupled matters are dried and placed on a gold label pad. Mouse anti-human IgM of the detection line is immobilized, and antigen antibody complexes of the gold label pad are movable. The kit further comprises the quality control system composed of one couple of an antigen and an antibody which can have specific reaction, wherein one of the couple is coupled with the colloidal gold and then placed on the gold label pad as a mobile phase; the other one is immobilized at a quality control line (C) as an immobile phase. The kit provided by the invention can assist in obtaining detection results in 2-15 minutes and can be conveniently applied to single specimen detection.
Description
Technical field
The present invention relates to external diagnosis reagent field, particularly, the present invention relates to a kind of colloidal gold chromatographic prize law and detect kit of the antihepatitis A virus IgM in human serum, body fluid or other sample and preparation method thereof.
Background technology
Viral hepatitis type A (is called for short hepatitis A, HA) be a kind of enteric infectious disease caused by hepatitis A virus (HAV), based on hepatic parenchymal cells inflammatory damage, infectiousness is strong, the incidence of disease is high, the outburst of food type and drinking water type and popular easily occurs, is still the public health problem that the whole world is general so far, whole world year number of the infected about 1,400,000, actual case is then 3 to 10 times of number of reports.It is the first that the incidence of disease of current China hepatitis A and infection rate still occupy other Various Types of Hepatitis, the Seroepidemiological study of viral hepatitis result display carried out the national beginning of the nineties, China's total prevalence rate of anti-HAV IgG is 80.9%, show the outburst of prevention and control hepatitis A with popular will be arduous long-term work, be also important research topic.
China's hepatitis A (hepatitis A) is main in distributing distribution, but time have and to break out or popular; 9.7 hundred million people are about had to infect HAV.China's hepatitis A epidemic characteristic has: 1, rural area is higher than city.5-15 year childhood infection rate city be starkly lower than rural area, but within more than 30 years old, to infect curve Urban and rural area be parastate.2, North of Yangtze River is higher than on the south the Changjiang river, west area higher than in, eastern region.3, show according to the distribution situation of each province and city prevalence rate, hepatitis A morbidity or eruption and prevalence are still based on children.4, prevalence rate is obviously relevant with hygienic habit.
1988, Shanghai hepatitis A was very popular, and continued nearly 1 year, involved number and reach 310,000.This event is because its number of the infected is many, impact dark, economic loss is heavy and make a stir all over the world.
Because hepatitis A complicated clinical manifestation is various, what have is even asymptomatic, therefore bring certain difficulty to diagnosis, not only will with reference to contact history, the sings and symptoms of the popular history of location hepatitis A, patient, but also liver function test and determination of serology will be made, the latter especially makes a definite diagnosis the foundation of hepatitis A.
Anti-HAV IgM is an important indicator of diagnosis viral hepatitis type A.Hepatitis A virus specific antibody mainly contains two kinds, i.e. the anti-HAV of IgM and IgG type.Anti-HAV IgM antibody occurs when acute Hepatitis A infection comparatively early (patient can detect HAV-IgM antibody in serum in 1 ~ 4 week after morbidity), rise very fast, peak titre is higher, titre is passed in time and declines, but by responsive immunoassay, the sustainable existence of IgM 6 ~ 12 months, even reaches 18 months.All can Anti-HAV IgM be found in the early stage body of all morbidity, and the index that anti-HAV IgG is Recovery Phase of diseases just to be occurred, be the protection antibody of viral hepatitis type A.Therefore, detecting anti-HAV IgM in patients serum is the method that current diagnosis hepatitis A is reliable the sensitiveest, this disease of can clarifying a diagnosis.
The method that can be used for anti-HAV IgM detection at present has radio-immunity (RIA), enzyme linked immunological (ELISA), chemiluminescence (CLIA) etc.These methods are laboratory method without exception, need certain specimen amount and detection time, detect while being generally used for a large amount of sample, after needing to collect a certain amount of sample, unification detects, and clinical practice generally needs within 1-5 days, send survey report.This is just unfavorable for emergency case and epiphytotics control.
Summary of the invention
An object of the present invention is to provide a kind of kit of quick detection antihepatitis A virus IgM.Detection kit provided by the present invention utilizes prize law reaction principle, and association colloid gold immunochromatography technique.Kit comprises anti-human IgM, the antigen antibody complex of colloidal gold conjugate and supportive material and Quality Control reactive system.
Wherein, described anti-human IgM can be goat-anti people IgM, also can be mouse-anti people IgM.
The antigen antibody complex structure of described colloidal gold conjugate is [collaurum]-[antibody]-[antigen], and wherein antibody is monoclonal or the polyclonal antibody of anti-hepatitis A virus.
Technical scheme of the present invention is coated on detection line (T) for adopting anti-human IgM.Virus antigen-antibody compound and colloidal gold conjugate drying are placed on gold and mark and pad.Wherein the mouse-anti people IgM of detection line is solidification, and the antigen antibody complex of gold mark pad is flowable.During test, sample is upwards flowed by chromatography effect, antibody in sample can be combined by the antigen-Gold Conjugate A on gold pad, and the anti-human IgM on tested measuring tape catches, [mouse-anti people IgM]-[serum antibody]-[Ag-Ab-collaurum] compound formed, thus show a red stripes at detection line (T).Negative sample in this way, then manifest redfree band.
Described supportive material comprises thieving paper, nitrocellulose filter, glass fibre, nonwoven fabrics, dacron, end card.Chromatography effect strengthened by thieving paper, and nitrocellulose filter is for the preparation of Stationary liquid, and glass fibre, nonwoven fabrics and dacron are that end card is then supportive backing for the preparation of mobile phase and absorption sample.
Conveniently carry out product Quality Control control, kit also comprises a quality control system, can the antigen-antibody of specific reaction form by a pair, and wherein one is placed on gold with colloidal gold conjugate and marks and pad, and is mobile phase.Another solidifies in nature controlling line (C), is Stationary liquid.When detecting, mobile phase dissolves backward upper chromatography, and is bonded on nature controlling line.
The antigen-antibody of described quality control system to mouse IgG-sheep anti mouse can be selected, wherein after mouse IgG coupling collaurum for mobile phase, sheep anti mouse be used for Stationary liquid.
Another object of the present invention is to provide a kind of preparation method of detection kit described above, comprises the following steps:
1. immobilon-p preparation: with damping fluid, detection line is drawn film liquid (mouse-anti people IgM) and be diluted to 1-2mg/ml,
Nature controlling line is drawn film liquid (sheep anti mouse) and be diluted to 2-4mg/ml, mark two waterlines by the concentration of 1 μ l/cm at the middle part of nitrocellulose filter.Nitrocellulose filter room temperature or 37 DEG C of drying for standby.
2. colloidal gold conjugate: by 5-10 μ g albumen: the rate of charge of 1ml collaurum by HAV antibody coupling on collaurum.The antibody that centrifugal segregation is unnecessary.Drop into HAV antigen in the ratio of 1: 10-1: 40 again, after room temperature reaction, the antigen that centrifugal segregation is unnecessary, obtains [collaurum-antibody-antigene] coupled complex.Be laid on by coupled complex on gold mark pad, 37 DEG C of drying for standby are detecting pad.
3. Quality Control mobile phase preparation: by 10-20 μ g albumen: Quality Control mobile phase albumen (mouse IgG) is coupled on collaurum by the rate of charge of 1ml collaurum, the albumen that centrifugal segregation is unnecessary, 37 DEG C of drying for standby are Quality Control pad.
4. paste the large plate of preparation: upper thieving paper, immobilon-p, detecting pad, Quality Control pad, sample pad are pasted onto successively on base plate.Base plate can also be pasted the adhesive sticker bar of protectiveness or index.
5. cutting little bar: by the width of 2.5-4mm, large plate is cut to little bar, is test strips.
6. optionally assemble plastics deck: be placed on by little bar in the plastics deck of upper and lower two panels, plastics deck has two windows at immobilon-p and sample pad place, is observation window and application of sample window.Test card is commonly referred to as after adding plastics deck.
7. single part of inner packing: be packaged in aluminium foil bag by test strips or test card, also needs in bag to add a slice drying agent, optionally adds disposable dropper.
In the method for immune detection antibody, one is envelope antigen, and the pattern resisted with enzyme or other indicant mark anti-human two, is called indirect method.One is that bag is resisted by anti-human two, by the pattern of enzyme or other indicant labelled antigen, is called prize law.Pattern of the present invention is prize law.
Kit of the present invention, can obtain testing result, and can be conveniently used in the detection of single part of sample in 2-15 minute.Its using value is that (1) emergency treatment is applied, and can judge the cause of disease rapidly.(2) speed that can be the fastest judges the popular of hepatitis A virus, and makes it to be effectively controlled.(3) basic medical unit that a lot of reported cases amount is less, needed sample to be sent to upper level unit, the thus delay diagnosis time originally.
New technology can make basic medical unit obtain detectability, thus earlier can diagnose the infection of hepatitis A virus.
Accompanying drawing explanation
The hierarchical diagram of Fig. 1 test card
The side view of Fig. 2 test strips
Wherein, (1) well (2) sample pad (3) Quality Control pad (4) detecting pad (5) immobilon-p (6) watch window (7) nature controlling line (C) (8) end card (9) upper thieving paper (10) plastic clip upper slice (11) plastic clip bottom sheet (12) detection line (T)
Fig. 3 paper box result decision method
Embodiment
Embodiment one: kit preparation technology
Kit preparation process comprises the following steps:
1. immobilon-p preparation;
2. colloidal gold conjugate;
3. Quality Control mobile phase preparation;
4. paste the large plate of preparation;
5. cut little bar;
6. optionally assemble plastics deck;
(1) immobilon-p preparation
1. detection line draws film liquid: dilute mouse-anti people IgM for 1-2mg/ml with 0.02M (PH=7.4) PB, mixing, with for subsequent use after 40 μm of membrane filtrations.
2. nature controlling line draws film liquid: dilute for 2-4mg/ml with 0.02M (PH=7.4) PB by sheep anti-mouse igg, mixing, with for subsequent use after 40 μm of membrane filtrations.
3. rule in the middle part of nitrocellulose filter with drawing film instrument.Drawing film parameters is: scribe head height 4.8cm, bow pen spacing 0.5-1cm, line concentration 0.8-1 μ l/cm, line speed: 100-150mm/s.
4. lower than under 35% humidity, 37 DEG C of dryings more than 3 hours.
(2) colloidal gold conjugate, produces detecting pad
1. use material
1.1 40-60nm diameter colloidal gold solutions
1.2 0.1M borate buffer (BB) (PH8.5-9.0)
1.3 10％BSA
1.4 eluents: 0.01M borate buffer (PH8.5-9.0), 0.5%BSA
1.5 gold medal mark dilutions: 0.01M borate buffer (PH8.5-9.0), 0.5%BSA, 1%Tween-20,2% sucrose
2. use clean special glass graduated cylinder to get colloidal gold solution 100ml, observe outward appearance red, transparent, without phenomenons such as precipitation and variable colors, it all poured in round-bottomed flask.In round-bottomed flask, add magnet rotor, put on magnetic stirring apparatus, open and stir and be adjusted to suitable rotational speed.Use pipettor to get 0.1MBB 10ml, observe appearance transparent, without phenomenons such as muddiness, precipitation and variable colors, then added in flask.
3. use pipettor to take Anti-HAV 0.5-1mg.Observe appearance transparent, without phenomenons such as muddiness, precipitation and variable colors, it all added in collaurum.Note not being added to flask inwall or spilling.Keep agitation 5 minutes.
4. use pipettor to get 10%BSA 5ml, observe outward appearance yellow transparent, without phenomenons such as muddiness, precipitation and variable colors, then it all added in flask.Keep agitation 5 minutes.Close and stir, gold is marked solution and divide equally in centrifuge tube, centrifugal 30 minutes of 10000-12000rpm, inhale and abandon supernatant.
5. merge each centrifuge tube, use pipettor to get HAVAg, sampling amount is determined according to raw material working concentration.Observe appearance transparent, without phenomenons such as muddiness, precipitation and variable colors, then added in above-mentioned vial.Suction nozzle is inhaled in the solution and is made a call to twice, to remove raffinate.
6. add gold mark eluent and return to 100ml, stirring at room temperature reacts 2 hours.Centrifugal 30 minutes of 10000-12000rpm, inhales and abandons supernatant.
7. each centrifuge tube adds gold mark eluent by original volume precipitation is redissolved, more centrifugal 30 minutes of 10000-12000rpm, and supernatant is abandoned in suction.Getting gold mark dilution adds in centrifuge tube, and precipitation is redissolved for 20-30% original volume.
8. conjugate is laid on glass fibre or polyester film, is placed in drying in oven and spends the night.Require that drying time reaches more than 16 hours under the condition of ambient air humidity lower than 35%.
9. the detecting pad of drying is cut into the slice of 4-5mm width.
(3) Quality Control mobile phase preparation, produces Quality Control pad
1. use material
1.1 40-60nm diameter colloidal gold solutions
0.1M borate buffer 1.2 (BB) (PH8.5-9.0)
1.3 10％BSA
1.4 eluent: 0.01M borate buffer (PH8.5-9.0), 0.5%BSA
1.5 gold medal mark dilutions: 0.01M borate buffer (PH8.5-9.0), 0.5%BSA,
1%Tween-20,2% sucrose
2. use clean special glass graduated cylinder to get colloidal gold solution 100ml, observe outward appearance red, transparent, without phenomenons such as precipitation and variable colors, it all poured in round-bottomed flask.In round-bottomed flask, add magnet rotor, put on magnetic stirring apparatus, open and stir and be adjusted to suitable rotational speed.Use pipettor to get 0.1MBB 10ml, observe appearance transparent, without phenomenons such as muddiness, precipitation and variable colors, then added in flask.
3. use pipettor to take mouse IgG 1-2mg.Observe appearance transparent, without phenomenons such as muddiness, precipitation and variable colors, it all added in collaurum.Note not being added to flask inwall or spilling.Keep agitation 5 minutes.
4. use pipettor to get 10%BSA 5ml, observe outward appearance yellow transparent, without phenomenons such as muddiness, precipitation and variable colors, then it all added in flask.Keep agitation 5 minutes.Close and stir, gold is marked solution and divide equally in centrifuge tube, centrifugal 30 minutes of 10000-12000rpm, inhale and abandon supernatant.
5. each centrifuge tube adds gold mark eluent by original volume precipitation is redissolved, more centrifugal 30 minutes of 10000-12000rpm, and supernatant is abandoned in suction.Getting gold mark dilution adds in centrifuge tube, and precipitation is redissolved for 20-30% original volume.
6. conjugate is laid on glass fibre or polyester film, is placed in drying in oven and spends the night.Require that drying time reaches more than 16 hours under the condition of ambient air humidity lower than 35%.
7. the Quality Control pad of drying is cut into the slice of 4-5mm width.
(4) the large plate of preparation is pasted;
1. operation site: drying room worktable, should confirm before preparation to clear out a gathering place in working site, dehumidifier opened and ambient humidity lower than 35%.
2. thieving paper is cut into the width of 3cm.
3. paste thieving paper onto the backing plate: require: thieving paper lower edge covers nitric acid film upper edge 1mm.
4. paste detecting pad onto the backing plate: require: the upper edge of golden paper is answered under coverlay along 1.5-2.0mm.
5. paste Quality Control pad onto the backing plate: require: the upper edge of golden paper should reach 1/2 place of detecting pad and press against lower edge.
6. paste sample pad onto the backing plate: require: the upper edge of sample pad must press against lower edge at 1/2 place of Quality Control pad.
7. optionally paste protectiveness or indicative adhesive sticker
(5) little bar is cut;
1. pair cutting cutter is debugged, and arranging slitting width is 3-4mm.
2. be put in draw-in groove place by smooth for kilocalorie, carry out slitting.
3. breakage, spot, the underproof little bar of mark are chosen.
4. fill aluminium foil bag.Require: every bag of a test paper, a drying agent.
5. lot number is beaten in sealing.
(6) optionally plastics deck is assembled;
1. little bar is put in the middle part of plastic clip base, cover upper cover, compress.
2. fill aluminium foil bag.Require: every bag of a test paper, a drying agent.
3. lot number is beaten in sealing.
Embodiment two: ELISA test strip operating process:
1. sample is done 1: 100 dilution.(get disposable test tube, first add 10 μ l samples, then add 1000 μ l physiological saline, mix).
2. open aluminium foil bag, taking-up test strips/block for subsequent use.
3. test strips: sample application zone is below MAX line.Test strips MAX arrow direction to be immersed in oneself 1: 100 diluted sample until mark line, guarantees that soak time is no less than 15 seconds, then by test strips horizontal on recording chart (available A4 paper replaces), start timing simultaneously.
4. test card: the sample drawing oneself 1: 100 dilution of 100 μ l, adds in the well of test card.
5. can observe red liquid to flow to upper end, and be deposited as red line at the area of observation coverage.
6. test result should judge in 15 minutes.It is invalid to judge after 15 minutes.
Embodiment three: result judges
1. the positive (+): two red stripes occur.Article one, be positioned at detection line (T), another is positioned at nature controlling line (C).
2. feminine gender (-) a: only red stripes appears in nature controlling line (C), in detection line (T), redfree band occurs.
3. invalid: red stripes does not appear in nature controlling line (C), shows this invalidate the test.
Embodiment four: kit performance evaluation
1. the mensuration of limit of identification reference material
Through the inner quality control dish that National reference is demarcated, comprising positive reference material 10 parts, numbering P1-P10.Negative reference product 10 parts, numbering N1-N10.Limit of identification reference material 4 parts, numbering L1-L4.Wherein L1 is equivalent to National reference 1: 4, L2 and is equivalent to National reference 1: 8, L3 to be equivalent to National reference 1: 16, L4 be negative serum.Separately there is precision reference material 1 part.Inner quality control dish instructions requires that positive reference material and negative reference product recall rate are 100%, and limit of identification requires that L1, L2 should detect the positive, L3 can the moon can sun, L4 detects feminine gender, and precision reference material detects positive and colour developing is consistent.
We use the colloidal gold strip of three batches to measure Quality Control dish, and result is as shown in table 1.
Table 1: the measurement result of limit of identification reference material
| L1 | L2 | L3 | L4 | |
| 20090610 | + | + | ± | - |
| 20090620 | + | + | ± | - |
| 20090630 | + | + | ± | - |
Measurement result shows that the limit of identification of our test strips reaches L3, is equivalent to minimum output 1: 16 in National reference.The limit of identification of National reference respectively is 1: 2, and 1: 4,1: 8,1: 16, require that limit of identification reaches 1: 8, therefore, this kit can reach the requirement of respective country Panel.
2. analyze specificity assessment
Use the colloidal gold strip of three batches measure in inner quality control dish yin and yang attribute reference material, result is as shown in table 2.
Table 2: calibrating institute Quality Control dish yin and yang attribute reference material measurement result
Experiment shows, the specific performance of colloidal gold method meets the requirement of inner quality control dish.
3. cross reaction assessment
Collect other all kinds of C positives sample, use the colloidal gold method test strip of three batches to measure these serum samples, result as shown in Table 3-5.
Table 3:20090610 criticizes multiple virus-positive determination of serum result
| Sample classification | Total number | Negative number | Non-specific responding rate |
| C-hepatitis antibody is positive | 32 | 32 | 0％ |
| Hepatitis B surface antibody is positive | 64 | 64 | 0％ |
| Viral hepatitis type E IgM antibody is positive | 31 | 31 | 0％ |
| Anti-HBcIgM is positive | 10 | 10 | 0％ |
Table 4:20090620 criticizes multiple virus-positive determination of serum result
| Sample classification | Total number | Negative number | Non-specific responding rate |
| C-hepatitis antibody is positive | 32 | 32 | 0％ |
| Hepatitis B surface antibody is positive | 64 | 64 | 0％ |
| Viral hepatitis type E IgM antibody is positive | 31 | 31 | 0％ |
| Anti-HBcIgM is positive | 10 | 10 | 0％ |
Table 5:20090630 criticizes multiple virus-positive determination of serum result
| Sample classification | Total number | Negative number | Non-specific responding rate |
| C-hepatitis antibody is positive | 32 | 32 | 0％ |
| Hepatitis B surface antibody is positive | 64 | 64 | 0％ |
| Viral hepatitis type E IgM antibody is positive | 31 | 31 | 0％ |
| Anti-HBcIgM is positive | 10 | 10 | 0％ |
From above each table, the testing result of colloidal gold strip to every cross antigen is feminine gender, shows that its specificity is good, clinical diagnosis with other multiple virus, mistaken diagnosis can not occur.
4. the impact of haemoglobin
Different interpolation haemoglobin sample is measured by the test strips of lot number 20090610,20090620,20090630 batches, adding hemoglobin concentration is that reference done by the sample of 0mg/ml, all the other each sample colour developing situations compare with it, if there is false negative, positive sample colored intensity obviously reduces or background wash-out not exclusively causes detection line to be beyond recognition, then think that this concentration haemoglobin has interference to testing result.Three batches of test strips results are consistent.
Table 6: three batches of reagent detect the impact that different haemoglobin adds concentration sample
| Add concentration (mg/ml) | 0 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 12 |
| 10 parts of feminine genders | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 |
| 10 parts of positives | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 |
| L1 | + | + | + | + | + | + | + | + |
| L2 | + | + | + | + | + | + | ± | - |
| L3 | ± | ± | ± | ± | ± | ± | - | - |
| L4 | - | - | - | - | - | - | - | - |
After haemoglobin is added into 8mg/ml, detection zone wash-out is incomplete, and background obviously deepens, and sensitivity Quality Control colour developing shoals to being difficult to identification.
Clinical severe haemolysis hemoglobin concentration > 5mg/ml.Haemoglobin in view of 8mg/ml and above concentration measures anti-HAV IgM to this method and can cause false negative, therefore severe haemolysis sample can not use.
5. bilirubinic impact
Different interpolation cholerythrin sample is measured by the test strips of lot number 20090610,20090620,20090630 batches, adding bilirubin concentration is that reference done by the sample of 0mg/l, all the other each sample colour developing situations compare with it, if there is false negative, positive sample colored intensity obviously reduces or background wash-out not exclusively causes detection line to be beyond recognition, then think that this concentration cholerythrin has interference to testing result.The test paper measurement result of different batches is consistent, in table 7.
Table 7: three batches of test paper measure the impact that various biliary red pigment adds concentration sample
| Add concentration (mg/l) | 0 | 30 | 75 | 150 | 300 |
| 10 parts of feminine genders | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 |
| 10 parts of positives | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 |
| L1 | + | + | + | + | + |
| L2 | + | + | + | + | + |
| L3 | ± | ± | ± | ± | ± |
| L4 | - | - | - | - | - |
In normal human serum, total bilirubin (T.Bil) scope is 2.98 ~ 10.0mg/l, and severe jaundice patient scope is 200 ~ 300mg/l.Experiment confirms, when the bilirubin concentration in serum is up to 300mg/l, still significantly do not disturb the mensuration of Anti-HAV IgM, this content far exceeds the content of general jaundice patient, and therefore, jaundice serum sample does not affect substantially on this kit measurement.
6. the impact of cholesterol
Different interpolation cholesterol sample is measured by the test strips of lot number 20090610,20090620,20090630 batches, adding cholesterol concentration is that reference done by the sample of 0mg/l, all the other each sample colour developing situations compare with it, if there is false negative, positive sample colored intensity obviously reduces or background wash-out not exclusively causes detection line to be beyond recognition, then think that this concentration cholesterol has interference to testing result.The test paper measurement result of different batches is consistent, in table 8.
Table 8: three batches of test paper measure the impact that various biliary sterol adds concentration sample
| Add concentration (mg/ml) | 0 | 2 | 4 | 6 | 8 | 10 |
| 10 parts of feminine genders | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 |
| 10 parts of positives | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 | 10/10 |
| L1 | + | + | + | + | + | + |
| L2 | + | + | + | + | + | + |
| L3 | ± | ± | ± | ± | ± | ± |
| L4 | - | - | - | - | - | - |
Normal person's cholesterol scope 1.2-2.3mg/ml (3.1-5.95mmol/l), experimental result shows, serum cholesterol levels reaches 10g/L, colour developing does not have obvious tendency to change, this content far exceeds the content of general high fat of blood patient, therefore, cholesterol in serum is not disturbed this kit.
7. the impact of triglyceride
Mix by different proportion with normal human serum with non-hepatitis high fat of blood human serum, obtain variable concentrations triglyceride Serial serum, refer to table 9.Be used further to the different weak positive of preparation and negative sample.
Table 9 variable concentrations triglyceride serum is prepared
| Sequence number | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| High fat of blood human serum addition ml | 0 | 0.4 | 1.5 | 2 | 3 | 3.5 | 4 |
| Normal human serum addition ml | 4 | 4 | 3 | 2 | 1.5 | 1 | 0 |
| Triglyceride concentration mmol/l after mixing | 0.72 | 1.34 | 3.01 | 4.16 | 5.31 | 6.07 | 7.6 |
Different triglyceride content sample is measured by the test strips of lot number 20090610,20090620,20090630 batches, reference is done with No. 1 sample group (i.e. triglyceride concentration 0.72mmol/l), all the other each sample group colour developing situations compare with it, if there is false negative, positive sample colored intensity obviously reduces or background wash-out not exclusively causes detection line to be beyond recognition, then think that this concentration cholesterol has interference to testing result.Three batches of test paper performances are identical as a result.
Table 10: different batch reagent detects the impact that different triglyceride adds concentration sample
| Triglyceride concentration (mmol/l) | 0.72 | 1.34 | 3.01 | 4.16 | 5.31 | 6.07 | 7.6 |
| Normal person's sample | - | - | - | - | - | - | - |
| Strong positive sample | + | + | + | + | + | + | + |
| Weak positive sample | ± | ± | ± | ± | ± | ± | ± |
Triglyceride in serum normal high limit is 1.7mmol/L clinically, this experimental result shows, triglyceride in serum content does not have obvious tendency to change to colour developing during 7.6mmol/l, this content far exceeds the content of general high fat of blood patient, therefore, Triglycerides in Serum does not disturb this kit.
8. imprecision assessment
Use the colloidal gold method test strip of 20090610,20090620,20090630 batches, each batch of replication quality-control sample 10, result be positive and each batch in batch between colored intensity consistent, show reagent batch in imprecision and between criticizing imprecision better.
Claims (3)
1. detect a preparation method for the kit of antihepatitis A virus IgM fast, it is characterized in that, comprise the steps:
(1) immobilon-p preparation: with damping fluid, detection line is drawn film liquid mouse-anti people IgM and be diluted to 1-2mg/ml, nature controlling line is drawn film liquid and be diluted to 2-4mg/ml, two waterlines are marked at the middle part of nitrocellulose filter by the concentration of 1 μ l/cm, described damping fluid is the PB of pH value 7.4, it is mouse-anti people IgM solution that described detection line draws film liquid, and it is sheep anti mouse solution that described nature controlling line draws film liquid;
(2) colloidal gold conjugate: by 5-10 μ g albumen: the rate of charge of 1ml collaurum by HAV antibody coupling on collaurum, the antibody that centrifugal segregation is unnecessary, HAV antigen is dropped into again in the ratio of 1: 10-1: 40, after room temperature reaction, the antigen that centrifugal segregation is unnecessary, obtains collaurum-antibody-antigene coupled complex, is laid on by coupled complex on gold mark pad, 37 DEG C of drying for standby are detecting pad;
(3) Quality Control mobile phase preparation: by 10-20 μ g albumen: the rate of charge of 1ml collaurum by Quality Control mobile phase albumen coupling on collaurum, the albumen that centrifugal segregation is unnecessary, 37 DEG C of drying for standby, be Quality Control pad, described Quality Control mobile phase is mouse IgG;
(4) the large plate of preparation is pasted: upper thieving paper, immobilon-p, detecting pad, Quality Control pad, sample pad are pasted onto successively on base plate;
(5) cutting little bar: by the width of 2.5-4mm, large plate is cut to little bar, is test strips;
(6) be placed in the plastics deck of upper and lower two panels by little bar, plastics deck has two windows at immobilon-p and sample pad place, is observation window and application of sample window;
(7) single part inner packing: be packaged in aluminium foil bag by test strips or test card, also needs in bag to add a slice drying agent.
2. the preparation method of kit as claimed in claim 1, is characterized in that, the base plate described in step (4) is pasted the adhesive sticker bar of protectiveness or index.
3. detect a kit for antihepatitis A virus IgM fast, it is characterized in that, it is prepared from by the method described in claim 1 or 2.
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| CN201210125222.7A CN102692502B (en) | 2012-04-26 | 2012-04-26 | Rapid hepatitis A virus IgM antibody detection kit and preparation method thereof |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103336119B (en) * | 2013-06-27 | 2015-07-29 | 潍坊市康华生物技术有限公司 | Anti-HAV collaurum detects box |
| CN103364561A (en) * | 2013-07-29 | 2013-10-23 | 武汉中博生物股份有限公司 | Porcine circovirus 2 (PCV2) antibody colloidal gold immunity chromatography detection test paper and making method thereof |
| CN104330569B (en) * | 2014-10-29 | 2016-08-24 | 武汉生命科技股份有限公司 | A kind of test strips of quick detection antibody |
| CN110672850A (en) * | 2019-10-31 | 2020-01-10 | 迪瑞医疗科技股份有限公司 | Hepatitis A virus antibody IgM detection kit and preparation method thereof |
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Address after: 100076 Beijing city Fengtai District pan Temple No. 20 Patentee after: BEIJING NORTH INSTITUTE OF BIOLOGICAL TECHNOLOGY Address before: 100076 Beijing city Fengtai District pan Temple No. 20 Patentee before: Beijing North Institute of Biological Technology |
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